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The Journal of Immunology, 2003, 171: 5287-5295.
Copyright © 2003 by The American Association of Immunologists

Tapasin Is a Facilitator, Not an Editor, of Class I MHC Peptide Binding 1

Angela L. Zarling2,*, Chance John Luckey2,*, Jarrod A. Marto3,{dagger}, Forest M. White3,{dagger}, Cynthia J. Brame3,{dagger}, Anne M. Evans{dagger}, Paul J. Lehner4,§, Peter Cresswell§, Jeffrey Shabanowitz{dagger}, Donald F. Hunt{dagger},{ddagger} and Victor H. Engelhard5,*

* Carter Immunology Center and Department of Microbiology, and Departments of {dagger} Chemistry and {ddagger} Pathology, University of Virginia, Charlottesville, VA 22908; and § Howard Hughes Medical Institute, Yale University School of Medicine, Section of Immunobiology, New Haven, CT 06510


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tapasin has been proposed to function as a peptide editor to displace lower affinity peptides and/or to favor the binding of high affinity peptides. Consistent with this, cell surface HLA-B8 molecules in tapasin-deficient cells were less stable and the peptide repertoire was substantially altered. However, the binding affinities of peptides expressed in the absence of tapasin were unexpectedly higher, not lower. The peptide repertoire from cells expressing soluble tapasin was similar in both appearance and affinity to that presented in the presence of full-length tapasin, but the HLA-B8 molecules showed altered cell surface stability characteristics. Similarly, the binding affinities of HLA-A*0201-associated peptides from tapasin+ and tapasin- cells were equivalent, although steady state HLA-A*0201 cell surface expression was decreased and the molecules demonstrated reduced cell surface stability on tapasin- cells. These data are inconsistent with a role for tapasin as a peptide editor. Instead, we propose that tapasin acts as a peptide facilitator. In this role, it stabilizes the peptide-free conformation of class I MHC molecules in the endoplasmic reticulum and thus increases the number and variety of peptides bound to class I MHC. Full-length tapasin then confers additional stability on class I MHC molecules that are already associated with peptides.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The MHC class I Ag-processing system provides a mechanism by which CD8+ T lymphocytes gain information about the proteins being made within cells. Newly generated proteins are cleaved in the cytosol by proteasomes or other cytosolic proteases to short peptides of 8–12 aa in length. The TAP then transports these peptides into the lumen of the endoplasmic reticulum (ER). 6 Once in the ER, the binding of peptides to newly synthesized, empty MHC class I molecules is facilitated by the action of several accessory proteins that collectively have been described as the peptide-loading complex (1). Peptide binding to the MHC-{beta}2-microglobulin ({beta}2m) heterodimer results in a confirmation change and subsequent release of the heterotrimer from the loading complex. These newly formed, stable MHC class I/peptide complexes then travel via the default secretory pathway to the cell surface, where they are available for recognition by the appropriate TCR.

The ER resident protein tapasin is a member of the peptide-loading complex, and plays a critical role in the efficient loading and surface expression of MHC class I molecules. Work with the tapasin-deficient cell line 721.220 and subsequently with tapasin knockout mice has generally shown that the MHC class I molecules in tapasin-deficient cells have reduced stability and expression at the cell surface (2, 3, 4, 5, 6, 7, 8, 9, 10, 11). Interestingly, the magnitude of this effect varies among individual MHC class I alleles (2, 8, 12). One might expect such variation to reflect a number of roles for tapasin in class I MHC folding. Indeed, there are now several proposed mechanisms for tapasin function (13, 14).

When it was demonstrated that tapasin bound both TAP and empty MHC class I molecules simultaneously (10, 15), it was hypothesized that tapasin’s enhancement of MHC class I surface expression was due to its ability to localize newly synthesized empty H chain/{beta}2m dimers at the site of peptide entry into the ER and thereby to facilitate peptide loading. However, this simple idea was called into question by the observation that a soluble tapasin molecule, lacking the transmembrane and cytoplasmic regions that interact with TAP, restored MHC class I surface expression and Ag presentation in tapasin-deficient cells (11). It has also been shown that tapasin directly stabilizes TAP molecules in the ER membrane (9, 11, 13, 16). Thus, tapasin might augment MHC class I expression by increasing peptide availability in the ER via TAP. Finally, cell surface MHC class I molecules expressed in the absence of tapasin are less stable. This has been proposed to reflect the binding of lower affinity peptides, and has led to the suggestion that tapasin functions as a peptide editor to displace lower affinity peptides and/or to favor the binding of peptides with higher affinities (3, 6, 8, 9, 12, 13, 14, 17, 18). However, direct evidence that tapasin functions as a peptide editor to augment the presentation of high affinity peptides has not been directly demonstrated to date.

In the present study, we compared the peptides associated with HLA-B8 expressed on cells lacking tapasin with those expressed on cells encoding either a soluble or full-length tapasin protein. We chose HLA-B8 because its cell surface expression level is partially dependent on tapasin expression (2). In addition, previous work with the soluble tapasin construct had shown that soluble tapasin increases cell surface HLA-B8 levels to the same extent as full-length tapasin, but does not associate with TAP or up-regulate its expression (11). By evaluating HLA-B8 stability and the repertoire and binding affinity of HLA-B8-associated peptides in the presence and absence of tapasin, we conclude that tapasin does not function to augment the presentation of high affinity peptides. Instead, our results suggest that tapasin stabilizes class I MHC molecules both before and after peptide binding, and enables presentation of a broader peptide repertoire.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and Abs

The tapasin-deficient cell line 721.220.B8 (2) and its stable tapasin-transfected derivatives have been previously described (11). All cell lines were maintained at 37°C with 5% CO2 in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, and 300 µg/ml G418. The full-length or soluble tapasin-transfected .220.B8 cell lines were also grown under selection with 500 ng/ml puromycin. The tapasin-sufficient cell line 721.53 (HLA-A*0201+) was maintained in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, and HEPES, while the tapasin-deficient, HLA-A2-transfected cell line 721.220.A2 was maintained in RPMI 1640 medium supplemented with 5% FCS/SerXtend, 2 mM L-glutamine, and 300 µg/ml G418. SFR8-B6, a rat mAb that recognizes the Bw6 public Ag that HLA-B8 molecules express (19); W6/32, a {beta}2m-dependent monomorphic anti-human MHC class I mAb (20); and BB7.2, a mouse mAb specific for HLA-A2 (21), were used in this study.

HLA-B8 cell surface expression and stability

The cell surface stability of HLA-B8 molecules was measured by incubation of cells at 37°C in the presence of 10 µg/ml brefeldin A (BFA; Sigma-Aldrich, St. Louis, MO). At various times subsequent to addition of BFA, cells were harvested, washed, and resuspended in PBS containing 0.5% BSA and 0.2% sodium azide (flow cytometry staining buffer). Cells were processed for flow cytometry by incubation for 30 min at 4°C with SFR8-B6, washing three times, and incubation for 30 min with FITC-labeled mouse anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were subsequently washed three times with flow cytometry staining buffer, fixed with 0.5% paraformaldehyde, and analyzed on a BD Biosciences FACScan (Mountain View, CA). Live cells were gated, 20,000 events were counted, and the mean fluorescence intensity was recorded.

Steady state levels of either HLA-A*0201 or HLA-B8 were determined by staining in flow cytometry staining buffer, as described above, with BB7.2 mAb, followed by FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) or SFR8-B6 mAb, followed by FITC-labeled mouse anti-rat IgG, respectively.

The kinetics of expression of newly formed HLA-B8 molecules was measured after denaturation of cell surface HLA-B8 molecules with acid, as described (22). Cells were resuspended in 50 µl of 300 mM glycine (pH 2.5)/1% (w/v) BSA (acid wash) and incubated 3 min at 37°C. The suspension was neutralized with 100 µl of RPMI 1640 supplemented with 10% FBS, 0.5 N NaOH, and 0.2 M HEPES and centrifuged. Cells were resuspended in 200 µl of cell medium in the presence or absence of 10 µg/ml BFA and incubated for 4–8 h at 37°C. HLA-B8 expression was quantitated by flow cytometry, as described above. For BFA pretreatment, cells were resuspended in 10 µg/ml BFA and incubated at 37°C for 2 h. The cells were then centrifuged and treated, as described above.

Isolation of HLA-B8- and HLA-A2-associated peptides

HLA-B8 and HLA-A2 molecules were isolated, as described previously (22). Briefly, cells were lysed in 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Roche Diagnostics, Indianapolis, IN), 20 mM Tris-HCl, pH 8.0, 100 µM iodoacetamide, 5 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 5 mM EDTA, 0.04% sodium azide, and 1 mM PMSF (lysis buffer). HLA-B8 molecules were immunoaffinity purified using recombinant protein A-Sepharose beads (Pharmacia, Piscataway, NJ) that had been saturated either with the HLA-B8-binding mAb SFR8-B6 or W6/32 mAb and HLA-A2 molecules were immunoaffinity purified with beads saturated with the HLA-A2-binding mAb BB7.2. Peptides were separated using ULTRA-free-MC 5000 NMWL filter units (Millipore, Bedford, MA), and elution was completed into Teflon tubes (Savillex, Minnetonka, MN).

Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) data acquisition

For all MS and MS/MS analyses, appropriate aliquots of immunoaffinity-purified MHC-associated peptides were loaded onto C18 microcapillary HPLC columns with integrated electrospray emitters (23) and gradient eluted directly into the mass spectrometer. The three-dimensional differential display data were acquired on a home-built Fourier transform ion cyclotron resonance mass spectrometer (FTMS) (23); full scan liquid chromatography (LC)-MS spectra were collected at a rate of 1 scan/s. Aliquots containing 6 x 105 cell equivalents of immunoaffinity-purified HLA-B8-associated peptides were analyzed from tapasin-deficient or tapasin-sufficient samples. Data from LC-FTMS experiments were normalized and displayed as three-dimensional plots of chromatographic elution time (min) vs mass-to-charge ratio (m/z), and ion abundance (spot density) (22).

LC-MS data were also acquired on a triple quadrupole mass spectrometer (TSQ-7000; ThermoFinnigan, San Jose, CA) to estimate total peptide content for HLA-B8 and on a LCQ Deca mass spectrometer (ThermoFinnigan) for HLA-A2. Total ion chromatograms from these experiments were integrated over time using specific early and late-eluting peptides, common to all samples, as reference markers. A linear calibration curve was fit to data from aliquots of 1 x 103 to 1 x 106 cell equivalents of immunoaffinity-purified HLA-B8-associated peptides and 1 x 105 to 3 x 106 cell equivalents of immunoaffinity-purified HLA-A2-associated peptides, extracted from cells expressing full-length tapasin. Integrated total ion chromatograms obtained from peptide aliquots extracted from cells expressing either soluble tapasin or no tapasin were compared with the calibration curve as a measure of normalized cell equivalents. These numbers were used for appropriate dilution factors in subsequent peptide-binding affinity studies (see below).

Sequence information for HLA-B8-associated peptides immunoaffinity purified from tapasin-deficient or full-length tapasin-expressing cells was obtained via data-dependent LC-MS/MS acquisitions on a quadrupole ion trap mass spectrometer (LCQ; ThermoFinnigan). Analysis was performed on 5.4 x 106 cell equivalents. MS/MS data were searched against the nonredundant protein database maintained at the National Center for Biotechnology Information, using SEQUEST (24). Sequence assignments were verified by manual interpretation of the corresponding MS/MS spectra.

Equilibrium binding of peptides to purified HLA-B8 or HLA-A2

An equilibrium assay was used that measures the ability of test peptides to compete with a radiolabeled standard peptide for binding to purified MHC class I molecules (25, 26). Purified HLA-B8 molecules were incubated at room temperature with various amounts of the peptides eluted from .220.B8, .220.B8.soltapasin, or .220.B8.tapasin cells, together with the iodinated HLA-B8-restricted peptide FLKDY*QLL (*indicates radiolabel on Y) and 1 µM human {beta}2m (Calbiochem, La Jolla, CA) in PBS, pH 7.0, 0.05% IGEPAL CA-630 (Sigma-Aldrich), 1 mM PMSF, 1.3 mM 1,10-phenanthroline, 73 µM pepstatin A, 8 mM EDTA, and 200 µM N{alpha}-p-tosyl-L-lysine chloromethyl ketone. After 48 h, HLA-B8-peptide complexes were separated from free peptide by gel filtration, and both bound and free radiolabeled peptides were quantitated using a gamma counter. Likewise, purified HLA-A2 molecules were incubated with the iodinated HLA-A2-restricted peptide FLPSDY*FPSV, and various amounts of the peptides were eluted from 721.53 and 721.220.A2 cells, after quenching the acetic acid-containing extracts with 1 M Tris (~pH 11). Results shown are representative of at least two independent experiments for each set of extracted peptides. Each of the data points is the mean of triplicate determinations.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tapasin-dependent differences in the expression and stability of cell surface HLA-B8 molecules

To assess tapasin’s role in the expression and stability of MHC class I molecules, we evaluated HLA-B8 on the cell surface of a tapasin-deficient cell line, 721.220, that had been transfected with HLA-B8 (.220.B8) and either full-length tapasin (.220.B8.tapasin) or a tapasin molecule that lacks a membrane-binding domain and does not associate with TAP (.220.B8.soltapasin). As previously reported (11), cells transfected with either full-length or soluble tapasin expressed comparable steady-state levels of HLA-B8 at the cell surface, which were 4-fold higher than the level on cells expressing HLA-B8 alone (Fig. 1). We evaluated the influence of tapasin on the kinetics of expression of HLA-B8 molecules at the cell surface by briefly exposing the cells to dilute acid to remove surface MHC class I/peptide complexes, followed by incubation at 37°C for 4 or 8 h. In the absence of tapasin, acid treatment reduced the surface expression of HLA-B8 to an undetectable level (Fig. 2, A and B). Surprisingly, a small, but reproducible fraction (~6.5% in experiment shown in Fig. 2) of the HLA-B8 molecules on the surface of cells expressing full-length tapasin was resistant to denaturation with acid. Four hours after acid treatment, HLA-B8 surface expression in cells expressing full-length tapasin had recovered to ~65% of the pretreatment level, while in tapasin-deficient cells, recovery was only 40% of the pretreatment level. By 8 h postacid treatment, HLA-B8 expression in cells expressing full-length tapasin had almost completely recovered (95%), while the cell surface expression of HLA-B8 on tapasin-deficient cells was still only 65% of the pretreatment levels. This difference was even more apparent when all of the data were normalized to the steady state expression of HLA-B8 on cells expressing full-length tapasin (Fig. 2C), which enables a more direct comparison of the absolute number of molecules expressed at different times. These results demonstrate that tapasin promotes the egress of HLA-B8 molecules to the cell surface.



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  		FIGURE 1. Steady state cell surface expression of HLA-B8 molecules in the presence and absence of tapasin. Cells expressing HLA-B8 alone (.220.B8) or together with either full-length (.220.B8.tapasin) or soluble tapasin (.220.B8.soltapasin) were evaluated for cell surface expression of this molecule by flow cytometry using the HLA-Bw6-specific Ab SFR8-B6, as described in Materials and Methods. The dashed line indicates staining of .220.B8 cells with secondary FITC-conjugated rat anti-mouse IgG Ab alone.

 


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  		FIGURE 2. Re-expression of newly synthesized HLA-B8 molecules in the presence and absence of tapasin. The .220.B8, .220.B8.tapasin, and .220.B8.soltapasin cell lines were acid treated to remove steady state HLA-B8 molecules, as described in Materials and Methods, and incubated in cell medium alone or cell medium containing 10 µg/ml BFA for 4 or 8 h. HLA-B8 cell surface expression was determined by flow cytometry using the HLA-Bw6-specific Ab SFR8-B6. A separate aliquot of cells was acid stripped and stained immediately. Results shown are representative of seven independent experiments. A, HLA-B8 cell surface expression on tapasin-deficient .220.B8 or full-length tapasin-expressing .220.B8.tapasin cells before and after acid treatment and following 4-h incubation at 37°C in the absence or presence of 10 µg/ml BFA. The dashed line represents staining on acid-stripped cells with secondary FITC-conjugated mouse anti-rat IgG Ab only. B, HLA-B8 cell surface re-expression for each condition was normalized to the level of expression at T = 0 for each untreated cell line (the mean fluorescence intensity at T = 0 untreated set as 100%). C, The same data as in B, all normalized to the level detected on cells expressing full-length tapasin (.220.B8.tapasin) at T = 0 before acid treatment. D, Cell lines were pretreated with BFA (10 µg/ml) for 2 h before acid treatment, and then incubated in cell medium alone or cell medium containing 10 µg/ml BFA for 4 or 8 h. All data were normalized to the level detected on cells expressing full-length tapasin (.220.B8.tapasin) at T = 0 before acid treatment as in C.

 
We also evaluated the ability of BFA to block re-expression of newly synthesized molecules in the presence and absence of tapasin. As expected, HLA-B8 re-expression in tapasin-deficient cells was completely inhibited by BFA (Fig. 2, B and C). Surprisingly, in cells expressing full-length tapasin, ~10% of the initial cell surface HLA-B8 molecules was re-expressed in the presence of BFA. This is over and above the HLA-B8 molecules that were resistant to acid denaturation in these cells. Interestingly, this pool of BFA-resistant molecules appears to be limited, as the percentage of BFA-resistant HLA-B8 molecules on full-length tapasin-expressing cells did not increase between 4 and 8 h (Fig. 2, B and C). However, the expression of these acid-resistant, BFA-insensitive molecules could not be blocked by preincubation of the cells with BFA for 2 h before acid treatment (Fig. 2D), nor by incubation with NH4Cl (data not shown). All data in Figs. 1 and 2 were obtained using the HLA-Bw6-specific Ab SFR8-B6, but comparable results were also obtained using W6/32 (data not shown). These results demonstrate that tapasin promotes the formation of subsets of HLA-B8 molecules with enhanced resistance to acid denaturation and the effects of BFA.

We next determined whether soluble tapasin was comparable to full-length tapasin in its effect on HLA-B8 stability. In keeping with its ability to restore cell surface expression of HLA-B8 on .220.B8 cells (Fig. 1), soluble tapasin augmented the egress of these molecules measured after acid treatment (Fig. 2, B and C). Interestingly, however, the rate of HLA-B8 egress in the presence of soluble tapasin was slower than in the presence of the full-length form. In addition, the fraction of these molecules that survived acid denaturation (2.5%) was intermediate between that of molecules expressed in the presence and absence of full-length tapasin. Finally, after acid treatment and in the presence of BFA, an additional 5% of the HLA-B8 molecules in these cells was re-expressed in 4–8 h (Fig. 2B). Again, this value is intermediate between those of HLA-B8 molecules expressed in the absence and presence of full-length tapasin. These results demonstrate that soluble tapasin enhances egress of HLA-B8 molecules, confers resistance to acid denaturation, and facilitates migration of a fraction of HLA-B8 molecules to the cell surface in the presence of BFA. However, these latter properties are most evident in the presence of the full-length form of tapasin.

We also evaluated the stability of surface HLA-B8 complexes after incubation of cells at 37°C in the presence of BFA to block the egress of new complexes. HLA-B8 complexes expressed in the presence of full-length tapasin were remarkably stable, and showed only a 10–15% decay over an 8-h incubation period (Fig. 3, A and C). In contrast, ~50% of HLA-B8 molecules loaded in the absence of tapasin decayed during the same time. These differences were reproducible in five independent experiments. Because there is a pool of BFA-resistant molecules evident only in cells expressing full-length tapasin, we corrected these data by subtracting the percentage of BFA-resistant molecules re-expressed at 4 and 8 h after acid treatment (Fig. 3, B and D). Even after this correction, the HLA-B8 molecules expressed in the presence of tapasin were still more stable than those expressed in tapasin-deficient cells (Fig. 3, B and D). In five independent experiments, the stability of HLA-B8 molecules expressed in the presence of soluble tapasin was either comparable to that of HLA-B8 expressed in the presence of full-length tapasin (Fig. 3, A and B), or slightly lower (Fig. 3, C and D, and data not shown). Similar data were obtained using either SFR8-B6 or W6/32 (data not shown). Overall, these results demonstrate that HLA-B8 molecules expressed at the cell surface in the absence of tapasin are less stable than those expressed in presence of the full-length or soluble forms.



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  		FIGURE 3. Stability of cell surface HLA-B8 molecules in the presence and absence of tapasin. Cells expressing HLA-B8 alone (.220.B8) or together with either full-length (.220.B8.tapasin) or soluble tapasin (.220.B8.soltapasin) were incubated at 37°C in the presence of 10 µg/ml BFA for the indicated times. A and C, HLA-B8 cell surface expression at each time point was determined by flow cytometric analysis, as described in Materials and Methods, and normalized to the level of expression at time of BFA addition (the mean fluorescence intensity at T = 0 set as 100%). Two representative experiments are shown. B and D, Data in A and C adjusted to account for BFA-insensitive molecules expressed in the presence of tapasin. The percentage of BFA-resistant molecules was averaged from seven independent experiments and is for .220.B8, 1.5%; for .220.B8.soltapasin, 4.3%; and for .220.B8.tapasin, 9.14%.

 
The spectrum of peptides associated with HLA-B8 is altered by the presence of tapasin

To directly characterize the effect of tapasin on peptide selection, we used MS to compare the HLA-B8-associated peptides expressed on cells lacking tapasin with those expressed on cells encoding either a soluble or full-length tapasin protein. Peptides were extracted from affinity-purified HLA-B8 molecules, and peptide amount was estimated by integration of total ion chromatograms obtained from online microcapillary HPLC-MS analysis performed on a triple quadrupole mass spectrometer. This analysis indicated that the quantities of HLA-B8-associated peptides extracted from cells expressing either soluble or full-length tapasin were comparable, indicating similar levels of peptide occupancy of the HLA-B8 molecules (data not shown). The integrated total ion current of peptides extracted from tapasin-negative cells was ~4-fold lower than that of the other two cell lines, consistent with the 4-fold lower surface expression of HLA-B8. Similar results were obtained using either SFR8-B6 or W6/32 mAbs to purify the HLA-B8 molecules. This again indicates that these HLA-B8 molecules were similarly occupied with peptides, and also that there was not a disproportionate loss of HLA-B8-associated peptides from tapasin-negative cells during the extraction and purification process.

A more detailed comparison of these peptide extracts was made by using online microcapillary HPLC combined with FTMS. The high mass resolution, mass accuracy, extended dynamic range, and low chemical background of the FTMS facilitated direct comparison of entire peptide profiles, at the level of individual peptides, over a very wide range of peptide abundances. The HLA-B8-associated peptide profiles from cells expressing either full-length or soluble tapasin were very similar to one another, although some differences were discernible on close inspection (compare peptides in boxes in Fig. 4). In contrast, after normalization for the different quantities in each extract, the peptides expressed in the absence of tapasin were substantially distinct from those expressed in the presence of full-length tapasin (Fig. 5). Individual peptide sequences expressed in the absence or presence of tapasin were determined by MS/MS (Table I). Peptides expressed in the absence of tapasin still retained the major features of the peptide-binding motif for HLA-B8 ligands, consisting of a lysine or arginine at position 5 and a hydrophobic carboxyl terminus (27, 28, 29). In addition, many of the remaining peptides retained components of the minor anchors, including a lysine at position 3 or a proline or leucine at position 2. Several ligands were shared between the cells, regardless of the presence or absence of tapasin. Thus, the peptide repertoire associated with HLA-B8 in the absence of tapasin is markedly different from that presented in full-length tapasin-expressing cells, although the individual peptides retain important characteristics that determine binding to this MHC molecule.



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  		FIGURE 4. HLA-B8-associated peptide profiles from cells expressing full-length or soluble tapasin. HLA-B8 molecules from .220.B8 cells expressing full-length (.220.B8.tapasin, A) or soluble tapasin (.220.B8.soltapasin, B) were affinity purified using SFR8-B6 mAb, and the isolated peptides were analyzed by FTMS, as described in Materials and Methods. Equal numbers of cell equivalents of peptides were analyzed from each cell line. HPLC elution time is shown on the x-axis, mass/charge ratio on the y-axis, and the density of the spots corresponds to abundance of the peptide in the extract. The rectangles indicate regions in which the peptide profiles are similar, although there are slight instances of variation in expression or presence of a particular epitope presented by HLA-B8 on the two cell lines.

 


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  		FIGURE 5. The HLA-B8-associated peptide repertoire from cells deficient in tapasin is markedly different from that expressed in the presence of full-length tapasin. HLA-B8 molecules from cells expressing full-length tapasin (A) or from the tapasin-deficient .220.B8 cell line (B) were affinity purified using SFR8-B6 mAb, and the isolated peptides were analyzed by FTMS, as described in Materials and Methods. The data from the tapasin-deficient .220.B8 cell line are normalized to account for the 4-fold lower peptide yield obtained from these cells. The rounded rectangles indicate regions in which there is extensive diversity in the peptide profile derived from tapasin-deficient and full-length tapasin-expressing cells.

 

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  		Table I. Sequences of HLA-B8-associated peptides presented in the presence and/or absence of full-length tapasina

 
The MHC-binding affinities of peptides associated with HLA-B8 and HLA-A*0201 in the absence of tapasin are unexpectedly higher than those expressed in its presence

We next determined whether tapasin functioned to favor the binding of higher affinity peptides by testing the peptide mixtures purified from each of the three cell lines in a cell-free HLA-B8-binding assay. The relative average binding affinities of HLA-B8-associated peptides isolated from cells expressing soluble tapasin were almost identical with those extracted from cells expressing full-length tapasin (ratio of 1.0 ± 0.1) (Fig. 6A). Surprisingly, the average binding affinity of HLA-B8-associated peptides extracted from tapasin-negative cells was 3.9 ± 0.2-fold higher than that of the peptides from tapasin-positive cells (Fig. 6B). Thus, despite the fact that cell surface HLA-B8 molecules expressed in the absence of tapasin are less stable than those expressed in the presence of full-length or soluble tapasin, the peptides bound in the absence of tapasin have a higher HLA-B8-binding affinity.



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  		FIGURE 6. The peptides bound in the absence of tapasin have a much higher binding affinity than those expressed in the presence of full-length or soluble tapasin. Peptide mixtures purified from each of the three cell lines were tested in a cell-free MHC class I-binding assay, as described in Materials and Methods. Each data point is the mean ± SD of triplicate determinations. A and B, HLA-B8 molecules from cells expressing full-length (A, B) or soluble tapasin (A) or from the tapasin-deficient .220.B8 cell line (B) were affinity purified using SFR8-B6 mAb, and dilutions of the isolated peptide extracts were used to compete with a radiolabeled standard peptide for binding to purified HLA-B8 molecules. The extracts were compared based on the amount of peptide, as determined by MS. C, HLA-A2 molecules from cells expressing tapasin (721.53) or deficient for tapasin (721.220.A2) were affinity purified using BB7.2 mAb, and dilutions of the isolated peptide extracts were used to compete with a radiolabeled standard peptide for binding to purified HLA-A2 molecules. The relative amount of peptide was quantified by mass spectrometric analysis to determine the amount of HLA-A*0201-associated peptides isolated from tapasin-positive and tapasin-negative cells.

 
To determine whether this was generalizable, we compared the binding affinities of peptides presented by HLA-A*0201 in the presence or absence of tapasin. The steady state cell surface expression of HLA-A*0201 is ~1.5-fold lower on tapasin-deficient cells (data not shown) (2). HLA-A*0201 molecules were immunoprecipitated from either the tapasin-deficient, HLA-A*0201-transfected cell line 721.220.A2 or the tapasin-sufficient cell line 721.53 (HLA-A2+) using the HLA-A2-specific Ab BB7.2 (21). Mass spectrometric analysis revealed that the quantities of peptides extracted from cells deficient for tapasin were equivalent to that of tapasin-expressing cells (data not shown). When the pool of peptides was tested for its ability to bind to HLA-A*0201 in the equilibrium-binding assay, the HLA-A*0201-associated peptides extracted from tapasin-negative cells had equivalent average binding affinities to HLA-A*0201-associated peptides from tapasin-expressing cells (Fig. 6C). Thus, similar to what had been seen with HLA-B8 peptides, there is no evidence for binding of low affinity peptides in the absence of tapasin. Results with both HLA-A2 and HLA-B8 are inconsistent with a role for tapasin in augmenting the binding of higher affinity peptides.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this work, we have directly evaluated the repertoire and binding affinities of peptides associated with HLA-B8 molecules in the presence and absence of tapasin to better understand the role of tapasin in class I MHC folding and peptide association. HLA-B8 molecules expressed at the cell surface in the absence of tapasin were less stable than those expressed in its presence, with maximal stability depending upon coexpression of a full-length form of the tapasin molecule. In addition, the full-length form of tapasin optimally conferred resistance to acid denaturation and the ability to reach the cell surface in the presence of BFA to a fraction of HLA-B8 molecules. Use of HPLC and FTMS-based two-dimensional differential display revealed that the HLA-B8-associated peptides from cells expressing either full-length or soluble tapasin were very similar to one another, but significantly different from those expressed in the absence of tapasin. Based on all these characteristics, one would expect the peptides to be lower affinity in the absence of tapasin. However, the peptides bound in the absence of tapasin had a higher HLA-B8-binding affinity than those expressed in the presence of full-length or soluble tapasin. Similarly, the average binding affinities for the pool of HLA-A*0201-associated peptides from tapasin-positive and tapasin-negative cells were equivalent, although steady state HLA-A*0201 cell surface expression was decreased 1.5-fold and the molecules showed a slightly reduced stability on the surface of tapasin-negative cells. The high binding affinity of peptides on tapasin-negative cells cannot be accounted for by the loss of low affinity peptides during the extraction process, because the yield of peptides was in keeping with the cell surface MHC class I expression. Collectively, our results suggest that tapasin facilitates the binding of a more diverse set of HLA-B8-associated peptides by stabilizing a more receptive peptide-binding conformation, but does not function as a peptide editor to discriminate between low and high affinity peptides.

The cell surface expression of many human class I MHC molecules is reduced in the tapasin-deficient 721.220 cell line, although the magnitude of this effect varies among different alleles (2, 7, 8, 9, 10, 11, 18). Because TAP stabilization by tapasin does not influence cell surface expression in 721.220 cells, the loss of cell surface expression could result from a reduction in the folding or stability of class I MHC molecules inside the cell, or a reduction in the stability of cell surface molecules. In the present work, we have demonstrated that tapasin affects the stability of HLA-B8 molecules at the cell surface, and, based on their emergence in the presence of BFA, in intracellular compartments as well. Cell surface HLA-B8 molecules expressed in cells treated with BFA show a significantly lower stability in the absence of tapasin than in the presence of the full-length form. In addition, HLA-B8 molecules expressed in the absence of tapasin were completely denatured by brief exposure to acid, whereas a fraction of those expressed in the presence of the full-length form was acid resistant. However, the numbers of molecules of HLA-B8 re-expressed in 4 h after acid denaturation were substantially lower in the absence of tapasin than in its presence, and substantially lower than can be explained by the differences in cell surface stability. In addition, tapasin enabled a small fraction of HLA-B8 molecules to emerge at the surface in the presence of BFA. These results indicate that tapasin has an important effect on the stability of HLA-B8 molecules inside the cell.

Decreases in the stability of MHC class I molecules in the absence of tapasin are usually interpreted to reflect the binding of lower affinity peptides (3, 5, 6, 8, 9, 12, 17). The observation that the repertoire of peptides expressed in the absence of tapasin was substantially distinct from that expressed in its presence is consistent with this interpretation. Surprisingly, however, the HLA-B8-associated peptides isolated from cells deficient for tapasin had a 4-fold higher average binding affinity for HLA-B8 than the peptides from tapasin-positive cells. The yield of peptide from tapasin-deficient cells was consistent with the MHC class I cell surface expression and indicates that the MHC class I molecules were similarly occupied with peptide and that there was not a disproportionate loss of HLA-B8-associated peptides during the extraction and purification process from tapasin-deficient cells. Thus, although the HLA-B8 molecules formed in the absence of tapasin are less stable than those formed in the presence of the full-length form, the repertoire of peptides presented is of higher affinity. This observation challenges the idea that low stability of MHC class I molecules should be equated with binding of low affinity peptides. Instead, our data suggest that low stability may also be related to the conformational state of the MHC molecule after peptide binding. Consistent with this idea, HLA-A*0201-associated peptides from tapasin-negative cells have equivalent binding affinity to those from tapasin-positive cells, while the overall steady state levels of HLA-A*0201 cell surface expression are reduced. Taken together, these results suggest a model in which tapasin plays at least two roles. First, it broadens the spectrum of bound peptides, both in terms of complexity and of binding affinities. Second, it stabilizes the class I MHC molecule in a way that is not directly related to the affinity of the bound peptide.

Support for this model comes from an analysis of HLA-B8 molecules expressed in the presence of soluble tapasin, which does not interact with TAP, but restores cell surface expression to the same level as does full-length tapasin (11). The peptide repertoires from cells expressing either full-length or soluble tapasin were remarkably similar to one another, as were their HLA-B8-binding affinities. Thus, tapasin influences the peptide repertoire independent of a direct association with TAP. This demonstrates a direct role for tapasin in broadening the repertoire of peptides presented by HLA-B8 molecules. Despite this, soluble tapasin is less efficient than full-length tapasin in stabilizing HLA-B8 molecules against acid denaturation, and in enabling their egress in the presence of BFA. This suggests that full-length tapasin confers additional stability on HLA-B8 molecules that are already associated with peptides.

Previously published work is also consistent with these two effects of tapasin on class I MHC molecules. First, it has been shown that several tapasin variants, including one similar to the soluble tapasin used in the present work, failed to confer full stability on HLA-B*4402 molecules expressed in 721.220 cells (9). It was also demonstrated that these tapasin variants failed to fully induce a subset of Ab-defined epitopes on cell surface HLA-B*4402 molecules. Although these results were interpreted to reflect qualitative differences in the peptide cargo, peptide-binding affinities for HLA-B*4402 were not determined. In addition, MS comparisons, albeit of low resolution, suggested that the peptide repertoires were very similar. Second, HLA-B8 molecules formed in the presence of full-length tapasin were shown to mature more slowly compared with molecules formed in the presence of soluble tapasin (11). The present work establishes that this delay is not due to an influence of full-length tapasin on the peptide repertoire, and is instead consistent with the hypothesis that it is responsible for an additional quality control activity during prolonged ER retention.

Previous studies reported a high degree of similarity in the repertoire of peptides presented by HLA-A*0201, HLA-B*2705, and H-2Kb in the presence and absence of tapasin (6, 17, 18). Although these observations differ substantially from ours with HLA-B8-associated peptides, it should be noted that these groups used a relatively low resolution method (matrix-assisted laser/desorption/ionization time of flight MS), which may have obscured differences. In contrast, our binding data for HLA-A*0201-associated peptides are consistent with the idea that the peptides bound to this class I MHC molecule are not strongly influenced by the presence or absence of tapasin, despite the fact that it is less stable on the surface of tapasin-negative cells (4). Although the impact of tapasin on the repertoire of HLA-B8- and HLA-A*0201-associated peptides may differ, our data demonstrate that the reduced stability of both molecules in the absence of tapasin is not due to the presentation of low affinity peptides. Also, Purcell et al. (17) demonstrated that a small set of peptides selected from HLA-B*2705 in the presence and absence of tapasin showed a similar range of binding affinities, despite the reduced stability of this molecule in the absence of tapasin. In addition, the recovery of Kb-, HLA-B27-, and HLA-A2-associated peptides from tapasin-deficient cells was impaired, which was hypothesized to reflect the loss of low affinity peptides from the class I molecules (6, 17, 18). However, this observation could also be due to a lower stability of the class I MHC molecules in the absence of tapasin independent of the affinity of the bound peptides. In this regard, our rapid isolation methodology gave recoveries of peptides from tapasin-positive and tapasin-negative cells that were in keeping with the amount of class I molecules expressed, obviating any concern about selective loss of low affinity peptides. In addition, ours is the first study to examine the average affinity of the entire peptide mixture associated with a class I molecule in the presence and absence of tapasin.

By evaluating the binding affinity of peptides expressed in the presence and absence of tapasin, we have provided direct evidence that it does not function to optimize the binding of high affinity peptide ligands. Thus, it does not function as a peptide editor analogous to HLA-DM for MHC class II molecules (13, 14, 30, 31). Instead, we propose that one function of tapasin is to act as a peptide facilitator that increases both the number and variety of peptides bound to MHC class I. Because this leads to a lower average affinity of bound peptides, we suggest that tapasin accomplishes this by stabilizing the nascent class I molecule in the ER in an open, peptide-receptive conformation for a longer period of time. Tapasin might also act to stabilize a wider range of slightly different peptide-receptive conformations. This facilitator function may also occur in concert with other members of the peptide-loading complex. Thus, in the absence of tapasin, peptide competition for short-lived receptive class I MHC molecules favors those with higher average affinity or higher representation in the lumen of the ER. It is interesting to note that HLA-DM has been shown to stabilize open class II MHC molecules at low pH (13, 31, 32, 33), and thus does have a peptide facilitator function analogous to that proposed in this study, in addition to its editing function.

We propose that a second function of tapasin is to stabilize the peptide-class I MHC complex. The full-length form of tapasin contains all the elements necessary to confer the full stabilization of MHC class I molecules, many of which are compromised in several different tapasin variants as well as murine tapasin expressed in 721.220 cells (9). It is thereby linked to the ability of tapasin to interact with TAP, to efficiently participate in the peptide-loading complex, and to retain class I MHC molecules in the ER for a longer period of time. Although the exact mechanism is obscure, it seems likely that tapasin, acting independently or via other chaperonins in the loading complex, enables class I MHC-peptide complexes to fold more tightly. Interestingly, it was recently shown in an in vitro assembly system that the timing of TAP binding to peptide vs the tapasin-class I MHC molecule also influenced the thermal stability of the complex (34). Therefore, it may also be that the unique ability of full-length tapasin to stabilize class I MHC-peptide complexes is based on an ability to sense the status of TAP.

In summary, our work suggests a revision in the understanding of tapasin function is in order. Rather than editing the peptide repertoire to enhance the representation of high affinity ligands, tapasin broadens the repertoire to include a range of affinities, but also confers increased stability to these molecules. Further work is required to determine the basis for this stabilization function.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grants AI20963 (to V.H.E.) and AI33993 (to D.F.H.). A.L.Z. is a fellow of the Cancer Research Institute. C.J.L. was supported by Medical Scientist Training Program Grant GM07267. Back

2 A.L.Z. and C.J.L. contributed equally to this work. Back

3 Current address: MDS Proteomics, 1670 Discovery Drive, Suite 110, Charlottesville, VA 22911. Back

4 Current address: Cambridge Institute for Medical Research, Addenbrooke’s Hospital, Hills Road, Cambridge, U.K. Back

5 Address correspondence and reprint requests to Dr. Victor H. Engelhard, Carter Immunology Center and Department of Microbiology, University of Virginia, MR4 Box 801386, Charlottesville, VA 22908-1386. E-mail address: vhe{at}virginia.edu Back

6 Abbreviations used in this paper: ER, endoplasmic reticulum; {beta}2m, {beta}2-microglobulin; BFA, brefeldin A; FTMS, Fourier transform ion cyclotron resonance mass spectrometer; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry. Back

Received for publication June 10, 2003. Accepted for publication September 11, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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